Vibrio cholerae O1 causes the fatal epidemic disease cholera. The ability of V. cholerae to cause disease in humans is dependent upon two primary virulence factors, the toxin-coregulated pilus (TCP), a critical colonization factor, and cholera toxin (CT). The expression of these factors is controlled by a highly regulated transcriptional cascade that serves as a paradigm for the regulation of bacterial virulence. Expression of the cascade is initiated at the tcpPH promoter by a cooperative interaction between the regulators AphA and AphB. TcpPH and ToxRS are homologous pairs of transmembrane regulators that then cooperate to activate expression from the toxT promoter. ToxT, an AraC-type regulator, directly activates the expression of TCP and CT. Transcriptional activation of the virulence cascade is strongly dependent upon a variety of stimuli from the external environment. The long-term goals of this proposal are to understand the molecular basis of virulence gene regulation in V. cholerae so as to facilitate the development of new strategies to control its infectivity. Through a collaborative effort involving laboratories with expertise in structural biology, virulence gene regulation and pathogenesis, we have found that exogenous unsaturated fatty acids (UFAs), which are components of human bile, are capable of binding to ToxT and impairing its ability to activate virulence gene expression. UFAs bind into a ligand pocket in the N-terminal domain of ToxT and inhibit its dimerization as well as its ability to bind to DNA. Bicarbonate, which neutralizes the acid that comes from the stomach, has been shown to function as a second in vivo signal that, in contrast to UFAs, stimulates ToxT and enhances its DNA binding through an unknown mechanism. We have recently found in the current pandemic strain of V. cholerae, that in addition to influencing the activity of ToxT, exogenous UFAs also reduce the translation and stability of ToxT by different mechanisms through the master regulator of fatty acid (FA) metabolism, FadR. This proposal will build upon the ToxT structural and functional data, as well as our recent studies involving FadR, in order to elucidate several key mechanisms involved in regulating the expression of the virulence cascade. In Aim 1, we propose to elucidate the allosteric mechanisms controlling the dimerization of ToxT and to investigate the opposing effects of UFAs and bicarbonate on this process. In Aim 2, we propose to elucidate the different mechanisms by which FadR influences the translation and stability of ToxT. These studies will contribute significantly toward our understanding of how virulence gene expression is regulated in V. cholerae and will likely provide new avenues for antivirulence drug discovery.